Heme is essential to the function of nearly all cells and organisms, serving chiefly as a prosthetic group for oxygen-carrying proteins and enzymes involved in oxidation/reduction and electron transport reactions. Earlier work focused on in-depth studies on the regulation of, and inter- relationships between, the rate-controlling enzymes of hepatic heme synthesis (ALA synthase) and breakdown (heme oxygenase [HO])./ These studies helped to establish a central role of HO in physiologic heme breakdown and in regulation of activity of ALA synthase and the processes of hepatic porphyrin and heme metabolism. Excess heme may be toxic to cells, and the products of the HO reaction have physiologic roles, functioning as anti-oxidants (biliverdin, bilirubin) or as a neurotransmitter (carbon monoxide). Although two forms of HO have been identified, only form 1 (HO-1) is inducible by heme, certain other metalloporphyrins, and transition metals such as cadmium and cobalt. Metallothionein is another cellular protein involved in cellular defense against toxicity due to metals or other diverse stressful perturbations, and expression of the gene for metallothionein is also increased by these compounds. In previous work from this laboratory, HO-1 was purified from chick liver, and its cDNA was cloned and characterized. The regulation of expression f HO-1 has also been elucidated. The long term goal of this research program is to understand the regulation of hepatic porphyrin and heme metabolism and inter-relationships of this metabolism with cellular hemoproteins. To achieve this goal, the prior accomplishments will serve as the foundation for the achievement of the following specific aims; (1) Complete the sequencing and characterization of the gene for chick HO-1; (2) Carry out functional analysis of the 5'-flanking region of the chick HO-1 gene with the aid of transient and stable transfections of cultured cells using suitable reporter gene constructs; (3) Elucidate the molecular mechanism (s) whereby heme increases transcription of the HO-1 gene, focusing on causes for the transience of this induction and for the rapid development of refractoriness to induction; and (40 Compare the processes and mechanisms of induction of HO-1 and metallothionein mRNA's by heme with those produced by other inducers, particularly cadmium and cobalt and non- heme metalloporphyrins such as manganese- or tin-protoporphyrin. A major working hypothesis is that heme (and perhaps other metalloporphyrins) binds to protein(s0 that then interact selectively and specifically with key regulatory elements that control transcription and/or post-transcriptional activities of HO-1 mRNA. Because of the importance of heme metabolism and hemoproteins in the liver, the results of the proposed studies will provide fundamental new insights into normal biochemistry, molecular biology, and physiology and into key metabolic alterations that occur in disorders of heme metabolism, including the porphyrias, tyrosinemia, lead poisoning, and hyperbilirubinemia. The results will also provide new insights into the effects of transition metals or non-heme metalloporphyrin on normal hepatocytes. The latter are inhibitors of HO that, with heme, are finding increasingly important roles in medical therapeutics. Thus, greater understanding of factors that regulate levels of HO-1 and metallothionein are of broad importance in biology, medicine, pharmacology, and toxicology.